Objective: This study aims to explore the potential damage effect and related mechanisms of lansoprazole (LPZ) on rat cardiomyocytes (H9C2). Methods: After incubating H9C2 cells with 10 μmol/L LPZ for 0 h, 12 h, 24 h, and 48 h, DCFH-DA fluorescent probe was used to detect the level of ROS. And the expression of endoplasmic reticulum stress proteins (GRP78, CHOP) and apoptosis-related proteins (Cleaved-Caspase-12, Cleaved-Caspase-3, Cyt C, Bax/Bcl-2) was detected by western blot technique. In addition, the level of ROS, as well as the expression of endoplasmic reticulum stress and apoptosis-related proteins were evaluated after H9C2 cells incubating with LPZ alone or in combination with ROS inhibitor N-Acetyl-L-cysteine (NAC) for 48 h. Results: Lansoprazole could time-dependently increase the level of ROS, induce the expression of GRP78 and CHOP, and further increase the expression of Cleaved-Caspase-12, Cleaved-Caspase-3, Cyt C, and Bax/Bcl-2. NAC significantly reduced LPZ-induced ROS levels, and decreased the expression levels of GRP78, CHOP, Cleaved-Caspase-12, Cleaved-Caspase-3, Cyt C and Bax/Bcl-2. Conclusion: LPZ could induce cardiomyocyte apoptosis, and the mechanism may be related to oxidative stress and endoplasmic reticulum stress.